# US 12,111,310 B2 · Pretreatment agent in non-agglutination assays

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- Number: US 12,111,310 B2
- Document ID: US12111310B2
- Status: Granted
- Kind: B2
- Application number: 18/158,753
- Filed: 2023-01-24
- Published: 2023-05-25
- Granted: 2024-10-08
- Earliest claimed: 2013-12-13
- Assignees: Siemens Healthcare Diagnostics Inc.
- Applicants: Siemens Healthcare Diagnostics Inc.
- Inventors: Tie Q Wei; Christy Schaible
- Web page: https://patentgrep.com/patent/US12111310B2

## CPC

- G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding}
- G01N 33/9493: {Immunosupressants}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods and reagents are disclosed for minimizing a false result in an assay measurement for determining a concentration of an analyte in a sample suspected of containing the analyte. The method comprises pretreating both an antibody and a sample to be subjected to a non-agglutination immunoassay. In the method the antibody and the sample are combined with a pretreatment agent selected from the group consisting of hydroxyphenyl-substituted C1-C5 carboxylic acids and metallic salts thereof and halogen-substituted C1-C5 carboxylic acids and metallic salts thereof in an amount effective to enhance the accuracy of the non-agglutination immunoassay.

## Claims

### Claim 1

A method of determining an amount of an immunosuppressant drug present in a biological sample suspected of containing the immunosuppressant drug while minimizing inaccuracies resulting from interfering substances present in the biological sample, wherein the immunosuppressant drug is cyclosporine or everolimus, the method comprising a non-agglutination immunoassay comprising:
(a) combining, either simultaneously or wholly or partially sequentially, in an aqueous medium:
(i) the biological sample, wherein the biological sample has not been exposed to an extraction or separation step to extract or separate the analyte from other constituents of the biological sample;
(ii) a releasing agent for releasing the immunosuppressant drug from endogenous binding substances, wherein the releasing agent is a structural analog of cyclosporine when the immunosuppressant drug is cyclosporine, and wherein the releasing agent is a structural analog of everolimus when the releasing agent is everolimus, and wherein the releasing agent is present in the aqueous medium at a concentration in a range of from about 0.000001% to about 0.5%;
(iii) a pretreatment agent selected from the group consisting of hydroxyphenyl-substituted C1-C% carboxylic acids and metallic salts thereof and halogen-substituted C1-C5 carboxylic acids and metallic salts thereof in an amount effective to enhance the accuracy of the non-agglutination immunoassay by minimizing false results in an assay measurement caused by non-specific binding, wherein the pretreatment agent is present in the aqueous medium at a concentration in a range of from about 1% to about 3%;
(b) incubating the aqueous medium containing (i)-(iii) under conditions that allow for release of the immunosuppressant drug from endogenous binding proteins present in the biological sample;
(c) adding to the incubated medium containing (i)-(iii), either simultaneously or wholly or partially sequentially, competitive assay components (iv) and (v):
(iv) a competitive immunosuppressant drug reagent, wherein the competitive immunosuppressant drug reagent comprises the immunosuppressant drug or an analog thereof; and
(v) an antibody reagent comprising an antibody for the immunosuppressant drug, wherein the antibody specifically binds to the competitive immunosuppressant drug reagent but does not specifically bind to the structural analog of the immunosuppressant drug present in the releasing agent; and
wherein one of (iv) and (v) is immobilized and the other of (iv) and (v) is labeled;
(d) incubating the medium containing (i)-(v) under conditions that allow for binding of the antibody to immunosuppressant drug present in the biological sample or to the competitive immunosuppressant drug reagent, wherein the incubation temperature is 5° C. to 99° C. and the time period for the incubation is in a range of from about 0.2 seconds to about 24 hours;
(e) determining an amount of a complex formed of (iv) and (v) present in the incubated medium containing (i)-(v), wherein the amount of immunosuppressant drug present in the biological sample is inversely proportional to the amount of complex present.

### Claim 2 (depends on 1)

The method of claim 1, wherein when the immunosuppressant drug is cyclosporine, the structural analog of cyclosporine of (ii) is [Thr2, Leu5, D-Hiv8, Leu10]-cyclosporin A.

### Claim 3 (depends on 1)

The method of claim 1, and wherein the immunoassay by which the medium is examined is selected from the group consisting of enzyme immunoassay, a radioimmunoassay, an inhibition assay, an induced luminescence, a fluorescent oxygen channeling assay, and a fluorescence polarization assay.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the competitive immunosuppressant drug reagent of (iv) comprises an enzyme label, and the antibody reagent of (v) is immobilized on a magnetic particle.